The focus of our research program is to understand the structural basis of muscle contraction at the molecular level. The benefits of the research are not limited to understanding muscle contraction, but the contractile system of muscle can serve as a prototype for many other motile processes in living cells, e.g. cell division and nutrient transport. The basic processes of muscle contraction are well understood: it is a result of cyclic interactions between myosin and actin, driven by the energy of actomyosin ATP hydrolysis. Force is generated by myosin heads (cross-bridges) interacting cyclically with specific sites along the actin filaments. Shortening involves the relative sliding of the myosin filaments and the actin filaments. To explain the muscle movement, the working hypothesis is that some structural changes in the myosin molecule take place while interacting with actin. Since the availability of the crystal structures of the contractile proteins, and with the advent of single molecule assays, the field has made great strides in understanding the underlying processes. However, the details of the mechanism of transduction of chemical to mechanical energy still remains largely unresolved. One of the obstacles is that most of the studies at the molecular level are based on isolated, in vitro systems. The link between the information obtained from the in vitro systems and the actual processes occurring intact muscle is still largely missing. The aim of our efforts is to provide such a link. X-ray diffraction from permeabilized muscle cells, the technique used in the present study, is one of the few techniques that could reveal in vivo structures in living muscle cells, albeit at relatively low resolution. Crystal structures of myosin have revealed ligand-dependent differences (e.g. with ATP or ADP). It is critical to determine if such differences occur in a fully functioning muscle cell. Our results found that major characteristics of the myosin filaments in the muscle cells varied as a function of ligand bound in myosin as well as temperature. In fact, the state of the myosin filaments (ordered or disordered) is directly correlated with the atomic structures of myosin. Hence, a link as one of our goals between the microscopic conformation and the macroscopic filament structure has been established. Furthermore, x-ray diffraction patterns can provide a sensitive and relatively simple way of determining the distribution of myosin conformations in muscle cells. Accordingly, we have obtained preliminary results in understanding the mechanism of three small-molecule myosin ATPase inhibitors. These inhibitors, BDM, BTS, blebbistatin, greatly reduce the force level generated by the muscle. These small molecules appear to strongly stabilize (?trap?) one of the myosin conformations such that the ATPase cycle cannot be completed. Our newly initiated project in studying the structure of the cardiac muscle has been proceeding successfully with the goal to transfer our knowledge in the skeletal muscle to improve our understanding of the cardiac muscle. In FY04, we reported that the degree of weak cross-bridge binding, which has been shown to be obligatory for force generation in skeletal muscle, was modulated by the separation between thick and thin filaments in skinned cardiac muscle and thereby force is consequently modulated. This finding may play a role in explaining the Starling Law of the Heart. Structure of the contracting cardiac muscle will be initiated in FY05. In addition, the goal of the near future is to obtain information on the ligand dependency of the filament structure, as it was determined for the skeletal muscle.